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Kidney International, Vol. 64 (2003), pp. 2155–2162

VASCULAR BIOLOGY – HEMODYNAMICS – HYPERTENSION

Renin and kallikrein in connecting tubule of mouse ANDREAS ROHRWASSER,1 TOMOAKI ISHIGAMI,1 BARBU GOCIMAN, PIERRE LANTELME, TERRY MORGAN, TONG CHENG, ELAINE HILLAS, SHUHUA ZHANG, KENNETH WARD, MAY BLOCH-FAURE, PIERRE MENETON, and J.M. LALOUEL Department of Human Genetics, University of Utah, Health Sciences Center, Salt Lake City, Utah; Department of Human Genetics and University Claude Bernard Lyon I, Lyon, France; Department of Obstetrics and Gynecology, University of Utah, Salt Lake City, Utah; and Institute National de la Sant´ e et de la Recherche M´edicale U367, Paris, France

Renin and kallikrein in connecting tubule of mouse. Background. The observation of renin expression in connecting tubule, a segment that also expresses tissue kallikrein (KLK-1), raises two questions. Are the genes expressed in the same or in different cells of connecting tubule? Does this topography support the hypothesis that KLK-1 activates prorenin or is it more likely that it affords coordinated gene regulation? Methods. Renin and KLK-1 were examined by immunostaining and in situ hybridization. Renin activation by KLK-1 was investigated in vitro. In vivo, excretion of prorenin and active renin was compared in mice homozygous for targeted inactivation of KLK-1 (TK−/− ) and normal littermates (TK+/+ ). Results. Using in situ immunostaining for renin and in situ hybridization for KLK-1 mRNA, we found that connecting tubule cells expressing renin also expressed KLK-1. We confirmed in vitro activation of prorenin by KLK-1, but found no difference in the ratio of active renin to prorenin in urine of TK−/− and TK+/+ animals. Compared to TK+/+ controls, TK−/− mice exhibited significantly lower 24-hour excretion of prorenin (5.05 ± 1.16 mg Ang I/hour vs. 9.39 ± 1.96 mg Ang I/hour, P < 0.05) and active renin (1.98 ± 0.25 mg Ang I/hour vs. 3.58 ± 0.39 mg Ang I/hour, P < 0.05), with no difference in either urine volumes or plasma renin concentrations. Conclusion. Direct interaction between renin and KLK-1, not ruled out in vitro, is not supported in vivo. By contrast, lower excretion of active renin and prorenin in TK−/− compared to TK+/+ suggest coordinated regulation of the two proteins in their participation to collecting duct function.

Angiotensin II (Ang II) contributes to circulatory homeostasis by affecting both vascular tone and plasma fluid volume through multiple sites of formation and action [1]. The overlap in function and in distribution

1

These authors contributed equally to the study.

Key words: renin, prorenin, kallikrein, nephron, connecting tubule, urinary excretion, mouse. Received for publication March 18, 2003 and in revised form May 14, 2003, and June 18, 2003 Accepted for publication July 17, 2003  C

2003 by the International Society of Nephrology 2155

of Ang II originating from various sources has hampered a precise delineation of the relative contributions of systemic and local renin-angiotensin systems (RAS) to specific physiologic transactions. While the intrarenal effects of Ang II on sodium reabsorption as a function of dietary sodium have been recognized [2], the sites of formation and the mode of action of Ang II, as well as the regulation of its precursors, remain poorly understood [3]. A reexamination of the intrarenal distribution of renin and angiotensinogen in mouse has led to the delineation of a paracrine tubular RAS operating along the entire nephron [4]. Angiotensinogen synthesized in proximal tubule [5] is secreted into luminal fluid and transits through the entire nephron, as it can be recovered into final urine in uncleaved form in a manner dependent on acute changes in dietary sodium [4]. Furthermore, renin expression can be induced in a restricted nephron segment, the connecting tubule, by changes in either sodium intake or its fractional delivery in distal nephron segments [4]. Renin was observed in principal cells of the arcades formed by the connecting tubule of midcortical nephrons. Synthesis and secretion of tissue kallikrein (KLK-1) by connecting tubule has been well documented previously [6–8], and an inverse relationship has been reported between urinary kallikrein and essential hypertension [9]. In light of the closely interrelated functions of the RAS and the kallikrein-kinin system (KKS), including a possible role of KLK-1 in conversion of prorenin into active renin [10], an examination of the joint distribution of the two enzymes was indicated. This is particularly necessary in light of the well-documented cellular heterogeneity of the connecting tubule in rodents, as gradual transitions from distal convoluted tubule on the proximal side and to collecting tubule on the distal side lead to mixed cell populations [11, 12]. We show that principal cells of connecting tubule synthesize both enzymes, and that cells containing renin also exhibit kallikrein expression. We confirm that KLK-1 can activate recombinant

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Rohrwasser and Ishigami et al: Renin and kallikrein in connecting tubule

mouse prorenin in vitro, albeit at much higher concentrations that can be achieved with trypsin. This activation hypothesis was tested in vivo using transgenic mice homozygous for an inactivated KLK-1 gene (TK−/− ) [13] and their wild-type littermates (TK+/+ ). The observation of similar ratios of active renin to prorenin excretion in the two groups does not support a direct interaction between the two enzymes in intact animals. We did observe, however, a significant reduction in the excretion rates of both active renin and prorenin in TK−/− animals compared to controls, in the absence of significant differences in either plasma renin concentration or urine volume. The colocalization of the two enzymes in connecting tubule may afford coordinated regulation of renin and KLK-1 in terminal nephron. METHODS Animal studies All tissue studies were performed on C57BL/6 male mice. Examination of renin expression by in situ hybridization was performed following 24-hour water deprivation. All other tissue studies were performed in animals fed high sodium (3.15%) (Purina Mills, St. Louis, MO, USA) for 48 hours, with unrestricted access to food and water, and subjected to a subcutaneous amiloride injection (1 mg/kg). Kidneys were removed under anesthesia and processed as described below. For in vivo studies, six mice homozygous for an inactivated KLK-1 gene (TK−/− ) [13] and six wild-type (TK+/+ ) adult littermates were housed singly in metabolic cages (Nalgene, Rochester, NY, USA) for a 5-day habituation period under normal sodium diet and free access to water. Diet was substituted to high sodium for a 2-day period during which 24-hour urine samples were collected. Prior to the second 24-hour collection, animals received a subcutaneous injection of amiloride (1 mg/kg). All study protocols were approved by the Institutional Animal Care and Utilization Committees of the University of Utah and were in accordance with the French Guide for the Care and Use of Laboratory Animals. Immunohistochemistry Immunostaining was performed on thin serial sections (5 lm) prepared from paraffin-embedded kidneys. For the detection of renin, antibodies were raised against mouse submaxillary gland renin as described [4]; these antibodies recognize both renin and prorenin. Immunostaining was performed following a standard protocol (Dako Corp., Carpinteria, CA, USA). Briefly, paraffinembedded sections were deparaffinized and blocked in 3% blocking buffer (Roche Diagnostics, Indianapolis, IN, USA) and incubated overnight at 4◦ C in 1% blocking buffer in the presence of antimouse renin (1:200 dilution), antimouse H+ -adenosine triphosphatase (ATPase) (1:800 dilution), or control serum (1:400 dilution). Fol-

lowing washes, sections were incubated for 2 hours with 1:800 dilutions of biotinylated goat antirabbit IgG (Dako Corp.). The sections were labeled with alkaline phosphatase conjugated to streptavidine (30 minutes, 1:500 dilution, at room temperature). Immunostaining was visualized using nitroblue tetrazolium/5-bromo-4chloro3indoly-phosphate substrate (NBT/BCIP) (Sigma Chemical Co., St Louis, MO, USA). The sections were counterstained with Mayer’s hematoxylin or with periodic acid-Schiff (PAS), mounted, and photographed. In situ hybridization A 524 bp segment of the mouse KLK-1 gene was generated by performing reverse transcription-polymerase chain reaction (RT-PCR) on total RNA prepared from mouse kidney using the RNeasy system (Qiagen, Valencia, CA, USA). The intron-spanning primers were located in exons 2 and 3 and contained the Sp6 (forward→5′ ) and T7 (reverse→3′ ) promoter sequence. Digoxigeninlabeled sense and antisense probes were generated using 1 lg purified cDNA template, T7 or Sp6 RNA polymerase and the digoxigenin RNA labeling kit (Roche Diagnostics). Dilutions 1:10 and 1:100 of probes were prepared in hybridization buffer and used for hybridization. Paraffin-embedded sections from mouse kidneys were deparaffinized and dehydrated. The sections were digested with proteinase K (6 lg/mL for 30 minutes at room temperature), refixed briefly in 4% paraformaldehyde, and dehydrated. Negative control sections were RNase treated (RNase A; 40 lg/mL for 30 minutes at 37◦ C). Subsequent hybridization was performed following standard protocols using both dilutions of RNA probes. Following washes, digoxigenin was detected as described above using an alkaline phosphatase conjugated antibody (Dako Corp.). The sections were stained with NBT/BCIP, counterstained with Meyer’s hematoxylin or PAS, mounted, and photographed. Fluorescent activated cell sorting (FACS) analysis For proximal tubule and connecting tubule cell isolation, kidneys were removed from anesthetized animals, the capsule removed and the cortex dissected. Tissue sections were homogenized and further digested [0.5% collagenase, 0.1% DNase I, 0.01% soybean trypsin inhibitor (SBTI) 30 minutes, 37◦ C]. Cell suspensions were passed through a series of sieves ranging from 105 to 40 lm mesh size. Single cells were labeled with primary antibodies against calbindin-D28K (Swant, Bellinzona, Switzerland) or CD13 [fluorescein isothiocyanate (FITC)-conjugated rat antimouse CD13) (Research Diagnostics, Flanders, NJ, USA). Anticalbindin was detected with goat antirabbit secondary antibody conjugated with AlexaFluor 488 (Molecular Probes, Eugene, OR, USA). Cell suspensions were analyzed on a Becton Dickinson FACScan at 4◦ C and positive cells were collected. Pooled positive cells

Rohrwasser and Ishigami et al: Renin and kallikrein in connecting tubule

Table 1. Primer sequences Sequence 5′ to 3′

Primer Renin exon 5 5′ Renin exon 7 3′ GAPDH 5′ GAPDH 3′ KAP exon 1 5′ KAP exon 4 3′ KLK-1 5′ KLK-1 3′

TTG ACG GTG TCC TAG GCA TGG GCT CAG GGC TTG CAT GAT CAA CTT CAG TGG GAA GCT TGT CAT CAA CG ATG CAG GGA TGA TGT TCT GG CTT CTG TGG TCT GAC TGT GGC CTC ATC CAG GAT CAC TTC CTC GAT GCT GCA CCT CCT GTC C CAC AAC ATG TCA TCT GTC ACC

Abbreviations are: GADPH, glyceraldehyde-3-phosphate dehydrogenase; KAP, kidney androgen-regulated protein; KLK-1, tissue kallikrein; up, upstream primer; rp, reverse primer.

(∼50,000) were subjected to a second sort using the same parameters. Cells not labeled or only labeled with the secondary antibody served as negative controls. RT-PCR RNA from sorted cells was isolated using the RNeasy system (Qiagen). cDNA was generated by reverse transcription using oligo(dT)12−18 (Invitrogen, Carlsbad CA, USA) and Superscript reverse transcriptase (Invitrogen). Quantitative PCR analysis for glyceraldehyde3-phosphate dehydrogenase (GAPDH) was used to adjust cDNA amounts in all subsequent amplification reactions. PCR amplification were performed for KLK-1, kidney androgen-regulated protein (KAP), and renin with intron spanning primers (Table 1) and analyzed by gel electrophoresis. All PCR products were verified by sequencing. Water and genomic DNA served as controls in all amplification reactions. Preparation of prorenin-containing medium Stably transformed Chinese hamster ovary (CHO) cells expressing mouse renin (unpublished data) were grown to near confluence, washed three times in phosphate-buffered saline (PBS), and grown for another 16 to 24 hours in serum-free medium. The medium was collected, centrifuged, and filtered (0.22 lm syringe filter), and aliquots were snap-frozen in liquid nitrogen and stored at −80◦ C. Prorenin activation Prorenin-containing medium (10 lL) was incubated in activation buffer (50 mmol/L Tris-HCl, pH 8.0, and 150 mmol/L NaCl) with tosyl phenylalanyl chloromethyl-ketone (TPCK)-treated trypsin (Pierce, Rockford, IL, USA) or porcine pancreatic KLK-1 (Sigma Chemical Co.) in a 25 lL reaction. Trypsin or KLK-1 without prorenin, prorenin alone, and activation buffer served as negative controls. Activation and control reactions were performed at 37◦ C for 30 minutes. The activation reaction was stopped by the addition of 4-(2-aminoethyl)benzenesulfonyl-fluoride (AEBSF) (5 mmol/L) and ethylenediaminetetraacetic acid (EDTA) (5 mmol/L) and chilling on ice.

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Biochemical assays Plasma renin concentration was measured as described [14]. Blood was collected in heparin tubes and diluted (200 mmol/L Trizma base, 200 mmol/L maleate, and 40 mM EDTA, pH 8.5). Ten microliters were incubated with nephrectomized rat plasma for 1 hour at 37◦ C. The reactions were stopped on ice and diluted [100 mmol/L Tri-HCl, pH 8.5, 1 mmol/L EDTA, 0.5 g/L bovine serum albumin (BSA)] and the amount of generated angiotensin I (Ang I) measured by indirect radioimmuno assay (RIA) (NEN, DuPont, Boston, MA, USA). Renin activity in urine was measured as described previously [15]. Following renin activation, samples were diluted 1:50 and 5 lL aliquots were incubated for 10 minutes at 37◦ C with excess porcine angiotensinogen (2 lmol/L) (Sigma Chemical Co.) in a 25 lL reaction containing 50 mmol/L sodium-acetate (pH 6.5), AEBSF (1 mmol/L), 8-hydroxyquinoline (1 mmol/L), and EDTA (5 mmol/L). Highly purified mouse renin prepared from submaxillary gland [16] served as positive control, whereas buffer alone served as negative control. The reactions were stopped by boiling, and the formation of Ang I was measured in 10 lL aliquots using an indirect RIA (NEN). Renin activity was expressed as the amount of Ang I generated per hour. To activate urinary prorenin, 100 lL of urine were dialyzed against 50 mmol/L glycine-HCl buffer, pH 3.2, containing 150 mmol/L NaCl using a Microdialyzing System 100 (Pierce) for 24 hours with three buffer exchanges. After dialysis, each urine aliquot was carefully removed from the dialysis system and the volume measured. The pH of each sample was adjusted with 1 mol/L sodium phosphate buffer, pH 7, to a final pH of 7. For negative controls, 100 lL of urine were dialyzed against 50 mmol/L Tris-HCl buffer, pH 7, also containing 150 mmol/L NaCl and subsequently adjusted for volume with sodium phosphate. Renin activity was measured as described above. Dilution or concentration observed and measured during acid or control activations were used for final adjustment of renin activity. Urinary kallikrein activity was measured as described [13] and expressed as amount excreted per 24 hours. RESULTS Renin expression in connecting tubule In previous work, we documented renin immunostaining in connecting tubule of sodium-restricted mice [4]. Local synthesis could be demonstrated under conditions of acute sodium restriction by either in situ RT-PCR of kidney sections or by specific RT-PCR of mRNA extracted from pools of branched tubular segments [4]. Renin mRNA, although abundant in juxtaglomerular apparatus (JGA), could not be detected in tubular segments by direct in situ mRNA hybridization under these conditions. In the present work, this issue was revisited in

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Fig. 2. Colocalization of renin and tissue kallekrein (KLK-1) in connecting tubules of animals under high salt/amiloride conditions. KLK-1 mRNA (A) and renin immunostaining (B) are observed in the arcades formed by connecting tubules of midcortical nephrons. Signal is mutual exclusive in serial sections stained with either KLK-1 (C) and H+ -adenosine triphosphatase (ATPase) (D). Cells expressing kallikrein (arrowheads) do not stain for H+ -ATPase (arrows). Staining is concordant for KLK-1 (E) and renin (F ). Renin-positive cells also express KLK-1 (arrowheads), whereas cells not expressing KLK-1 (arrows) are also negative for immunoreactive renin. (A and B) 100× magnification. (C to F) 400× magnification. Counterstain is hematoxylin (A and B) or periodic acid-Schiff (PAS) (C to F).

Fig. 1. In situ hybridization of renin in mouse kidney after 24-hour water deprivation. At lower antisense probe concentration (1:100), specific staining is restricted to juxtaglomerular apparatus (JGA) cells (A). At higher antisense probe concentration (1:10), renin staining is also apparent in connecting tubule and proximal tubule (B). RNase treatment prior to hybridization eliminates all staining (inset). No significant staining is observed with the sense probe at high concentration (1:10) (C). (A to C) 100× magnification. Asterisks indicate JGA, arrows proximal tubules, and arrowheads connecting tubules. All sections are counterstained with periodicacid-Schiff (PAS).

animals subjected to 24-hour water deprivation. Again, expression was restricted to JGA when antisense probe was used at 1:100 dilution (Fig. 1A). At higher antisense probe concentration (1:10), renin mRNA became apparent in connecting tubule cells and, to a lesser extent, in proximal tubule (Fig. 1B). No staining was observed with high-sense probe concentration (Fig. 1C). Because greater sensitivity was achieved by immunohistochemistry, renin distribution was thereafter examined by immunostaining. Maneuvers such as water deprivation or sodium restriction, however, while activating connecting tubule renin, have the disadvantage of also leading to marked activation of JGA renin, leading to high renin concentration in vascular and interstitial fluid, with the potential of obscuring the detection of tubular renin. We have reported previously, however, that a single subcutaneous injection of amiloride (1 mg/kg) in animals subjected to an acute sodium load (see Methods

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Renin and kallikrein expression in sorted connecting tubule cells Connecting tubule cells were isolated after mechanical and enzymatic homogenization, followed by dual FACS sorting using calbindin-D28K , an established marker of connecting tubule cells (Fig. 3A) [18]. Proximal tubule cells were sorted using rat antimouse CD13, an aminopeptidase present in the brush border of this segment (Fig. 3B) [19]. Following FACS, RNA isolated

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Connecting tubule cells expressing renin also express KLK-1 Expression of KLK-1 in connecting tubule, under various conditions of dietary potassium and sodium intake, has long been established [17]. Whether renin and KLK-1 are expressed by the same tubular segments, and whether expression occurs in the same cells, as opposed to specialized subtypes, remained to be determined. Connecting tubule renin expression was induced by application of a high-salt/amiloride maneuver [4] (see Methods section) to C57BL/6 animals. Using immunostaining or in situ mRNA hybridization, KLK-1 was observed in cortical labyrinths, in clusters of tubular sections surrounding veins (Fig. 2A), a pattern characteristic of the arcades when examined in tissue sections. It was not observed in any other tubular segment of t...